Biology subjects

Sircar, D.

Publications and source records attributed to Sircar, D..

9 recordsLinked to original sources

bioRxiv2024

Seed specific overexpression of a modified wheat Or gene leads to enhanced β-carotene in rice and wheat grains

Vitamin A deficiency is a major public health problem affecting up to 50% of the worlds population, as either wheat or rice, which are poor in many essential micronutrients such as vitamin A, are major staple food crops. Biofortification of cereal crops with {beta}-carotene (provitamin A) through genetic engineering is a potential solution to overcome vitamin A deficiency. The Orange (Or) protein is involved in the regulation of carotenoid accumulation and previous studies demonstrated high carotenoid accumulation due to a single-nucleotide polymorphism (SNP) in the CDS leading to substitution of Arg to His in the OR protein results in carotenoid accumulation. In the present study, we showed that this substitution of a single amino acid at position 110 (Arg to His) of wild-type wheat TaOr (referred to as TaOrHis110) increased {beta}-carotene accumulation in transgenic wheat and rice plants overexpressing TaOrHis110 under the control of the seed-specific promoter Glu-1D1. HPLC analysis revealed increase in {beta}-carotene content in rice grain up to 8-fold in case of TP309 (japonica) cultivar, 13-fold in case of IET10364 (indica) cultivar and 7-fold in wheat cv. CPAN1676. Additionally, most of the carotenoid biosynthetic pathway genes were found to be upregulated in TaOrHis110 overexpressing seeds of TP309 and IET10364, which positively correlated with maximum increase in {beta}-carotene content.

plant biology↗

bioRxiv2024

Unveiling Mechanistic and Structural Insights of EstS1 Esterase: A Potent Broad-Spectrum Phthalate Diester Degrading Enzyme

The ubiquitous presence of plastics and plasticizers around the globe has raised an alarming condition. Phthalate diesters are high-priority pollutants that mimic natural hormones and act as endocrine disruptors upon entering living systems. While certain bacterial esterases have been identified for their role in phthalate diester degradation, their structural and mechanistic characteristics remain largely unexplored. A thermostable and pH-tolerant EstS1 esterase from Sulfobacillus acidophilus catalyzes the conversion of low molecular weight phthalate diesters to monoesters. This study highlights the unique potential of EstS1 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by employing biophysical and biochemical approaches along with in-depth structural analysis utilizing high-resolution crystal structures in both apo and complex forms, with various substrates, products, and their analogs to elucidate mechanistic details. The catalytic tunnel mediating entry and exit of the substrate and product, respectively, centralized the Ser-His-Asp triad performing catalysis by bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Additionally, structural analysis of the polypropylene analog jeffamine with EstS1 revealed effective covalent binding, demonstrating its multifunctional capability. Mutation analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore the potential of EstS1 as a key tool for advancing technologies aimed at phthalate diesters biodegradation.

biophysics↗

bioRxiv2024

Overexpression of barley heat stress transcription factor HvHsfA6a provide thermotolerance by thermopriming

Adverse impacts of climate change, including high temperature on cereal crop production, have been evidenced globally. In plants, heat shock factors (HSFs) are crucial components of heat stress associated rescue mechanisms and are also required for normal biological processes. Here, we functionally characterized a highly heat stress responsive HvHSFA6a in barley by developing constitutively overexpressing transgenic lines. These transgenic lines showed heat tolerant phenotype via improved photosynthesis, antioxidants and upregulation of HSPs and metabolites involved in stress amelioration and keeping thermomemory as compared to wild type plants. Global transcriptomics and ChIP sequencing revealed that HvHSFA6a orchestrates the expression of several genes through direct binding with other HSFs containing consensus HSE in their promoter regions. A GC-MS based metabolomics analysis also revealed the alterations in key metabolic processes such as carbohydrate metabolism, citric acid cycle, amino acids and secondary metabolism. Higher accumulation of key metabolites such as sucrose, galactinol, shikimate and ascorbate has been observed under both control and heat stress in transgenic lines as compared to wild type plants. Taken together, the results suggest that overexpression of HvHsfA6a prime the plants for heat stress conditions by alteration in gene expression and metabolic status. HighlightPriming is a mechanism by which plants respond to various abiotic and biotic stresses. Through multi omics approach we found that barley HsfA6a provide thermotolernce in transgenic plants through priming effect on transcriptome and metabolome.

molecular biology↗

bioRxiv2023

SmERF6 promotes the expression of terpenoid pathway in Salvia officinalis and improves the production of high value abietane diterpenes, carnosol and carnosic acid

Carnosol (CO) and carnosic acid (CA) are pharmaceutically important diterpenes predominantly produced in members of Lamiaceae, Salvia officinalis, Salvia fruticosa and Rosmarinus officinalis. Nevertheless, availability of these compounds in plant system is very low. With an effort to improve the in planta content of these diterpenes, SmERF6 (Salvia miltiorrhiza Ethylene Responsive Factor 6) transcription factor was expressed in S. officinalis heterologously. SmERF6 is known to bind at the promoter regions of Copalyl diphosphate synthase (CPS) and Kaurene synthase like (KSL) genes and improve ferruginol content, a common precursor for abietane diterpenes in Salvia genus. Transient expression of SmERF6 exhibited the inter-specific activity in promoting differential accumulation of diterpenes in S. officinalis. Overexpression studies showed elevation in the levels of CO (10-folds) and CA (8-folds). Further, in infiltrated leaves levels of ferruginol (50%) and CA derivatives (rosmanol, epirosmanal, methyl carnosic acid) were significantly upregulated along with the other signature terpenes. While, knockdown of homologous ERF6 resulted in drastic reduction of the metabolite content. Finally, stable transgenic lines of S. officinalis developed through in planta Agrobacterium mediated genetic transformation method accumulated higher levels of CO (4-folds) and CA (3-folds) as compared to wild plants. Overall, the present study is the first report on improving the content of pharmaceutically important diterpenes in S. officinalis by overexpressing pathway specific transcription factor. The current results showed convincing evidence for the concept of improving the content of specialized metabolite(s) in medicinal plants by manipulating the expression of key transcription factors.

plant biology↗

bioRxiv2022

Chloroplast activity provides in vitro regeneration capability in contrasting cultivars

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. We know little about why only some cultivars in crop plants are tissue culture friendly. In this study, tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency. Multiomics studies involving transcriptomics, proteomics, metabolomics, and biochemical analysis were performed using GP and D91 callus to unravel the regulatory mechanisms. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MSMS revealed 3062 protein groups and 16989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis also revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91. HighlightsMulti omics analysis revealed chloroplast play crucial role in providing in vitro regeneration capability in contrasting genotypes

plant biology↗

bioRxiv2022

Elucidation of the antiviral mechanism of natural therapeutic molecules Herbacetin and Caffeic acid phenethyl ester against Chikungunya and Dengue virus

Chikungunya (CHIKV) and dengue (DENV) viruses pose a public health risk and lack antiviral treatment. Structure-based virtual screening of natural MTase substrates library identified herbacetin (HC) and caffeic acid phenethyl ester (CAPE) as potential CHIKV nsP1 and DENV NS5 MTase inhibitors. Binding affinities and MTase inhibition were confirmed using purified proteins. Crystal structure of DENV3 NS5 MTase and CAPE complex revealed CAPE binding at GTP and cap 0 RNA sites. Interestingly, HC and CAPE depleted polyamines, which are crucial for RNA virus replication, and effectively diminished replication with IC50 values of [~]13.44 {micro}M and [~]0.57 {micro}M against CHIKV, and [~]7.24 {micro}M and [~]1.01 {micro}M against DENV, respectively. Polyamine addition did not reverse the antiviral effects, suggesting a dual inhibition mechanism. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/494145v5_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1f579eforg.highwire.dtl.DTLVardef@11862eborg.highwire.dtl.DTLVardef@67276org.highwire.dtl.DTLVardef@144c7f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

ROS/RNS balancing, aerobic fermentation regulation and cell cycle control a complex early trait ('CoV-MAC-TED') for combating SARS-CoV-2-induced cell reprogramming

In a perspective entitled From plant survival under severe stress to anti-viral human defense we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named ReprogVirus was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the ReprogVirus platform was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to RegroVirus complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called CoV-MAC-TED. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target CoV-MAC-TED in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that de-stressing disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.

cell biology↗

bioRxiv2021

Adaptive reprogramming during early seed germination requires temporarily enhanced fermentation a critical role for alternative oxidase (AOX) regulation that concerns also microbiota effectiveness

Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of plants inner and outer environment. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at systems levels. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as, germination, root and shoot growth, formation of adventitious, clonal and reproductive organs, fruits and seeds. Fermentative paths can help in acclimation and, to our view the role of alternative oxidase (AOX) in coordinating complex metabolic and physiologic adjustments is underestimated. Cellular level of sucrose is an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 hours after imbibition (HAI) was beneficial to germination. However, parallel enhanced AOX expression was essential to control negative effects by prolonged sucrose treatment. Early down-regulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose, but suppressed early germination in its presence. Our results also suggest that seeds-inoculated arbuscular mycorrhizal fungi can buffer sucrose stress during germination to restore normal respiration more efficiently. Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early selection on disease tolerance, predicting plant holobiont behavior and improving germination. Furthermore, our research strengthens the view that AOX can serve as powerful functional marker source for seed hologenomes.

cell biology↗

bioRxiv2021

Auxin-responsive (phospho)proteome analysis reveals regulation of cell cycle and ethylene signaling during rice crown root development

The rice root system, which primarily consists of adventitious/crown roots (AR/CR) developed from the coleoptile base, is an excellent model system for studying shoot-to-root trans-differentiation process. We reveal global changes in protein and metabolite abundance, and protein phosphorylation in response to an auxin stimulus during CR development. Global proteome and metabolome analyses of developing crown root primordia (CRP) and emerged CRs uncovered that the biological processes associated with chromatin conformational change, gene expression, and cell cycle were translationally regulated by auxin signaling. Spatial gene expression pattern analysis of differentially abundant proteins disclosed their stage-specific dynamic expression pattern during CRP development. Further, our tempo-spatial gene expression and functional analyses revealed that auxin creates a regulatory feedback module during CRP development and activates ethylene biosynthesis exclusively during CRP initiation. Ethylene signaling promotes CR formation by repressing the cytokinin response regulator, OsRR2. Additionally, the (phospho)proteome analysis identified differential phosphorylation of the Cyclin-dependent kinase G-2 (OsCDKG;2), and cell wall proteins, in response to auxin signaling, suggesting that auxin-dependent phosphorylation may be required for cell cycle activation, and cell wall synthesis during root organogenesis. Thus, our study provides evidence for the translational and post-translational regulation during CRP trans-differentiation downstream of the auxin signaling pathway. HighlightGlobal (phospho)proteome and metabolic profiling of rice CRP and CRs uncover differential proteins and metabolites associated with gene expression, cell cycle, ethylene signaling and cell wall synthesis during CR development.

plant biology↗